This invention relates to a method for producing N-myristoylated protein and, more particularly, to the co-expression of N-myristoyltransferase (NMT) and its protein substrates in E. coli.
Fatty acid acylation of specific eukaryotic proteins is a well established process which can conveniently be divided into two categories. On the one hand, palmitate (C16:0) is linked to membrane proteins via ester or thioester linkage post-translationally.
On the other hand, it is known that myristate (C14:0) becomes covalently bound to soluble and membrane proteins via amide linkage. This is believed to be a co-translational event. In the N-myristoylated proteins, amino-terminal glycine residues are known to be the site of acylation. Myristoyl CoA: protein N-myristoyltransferase (NMT, E.C. 2.3.1.97) catalyzes this co-translational modification. The NMT structural gene (NMT1) has recently been cloned from Saccharomyces cerevisiae. See Duronio et al., Science 243, 796-800 (1989). This gene encodes a polypeptide of 455 amino acids (M.sub.r =52,837).
A variety of viral and cellular proteins have been shown to be thus modified by the covalent attachment of myristate linked through an amide bond to glycine at their amino termini. Such modification is essential for the full expression of the biological function of some N-myristoylated proteins. An example of a most thoroughly studied myristoylated protein is the transforming protein of Rous sarcoma virus, p60.sup.v-src. Without the covalent attachment of myristate to its N-terminal glycine, the protein cannot transform cells even though its tyrosine kinase activity remains intact.
The myristoylation reaction can be represented as follows: ##STR1##
Further background information on the above protein fatty acid acylation can be had by reference to the following series of articles by scientists associated with the Washington University School of Medicine:
Towler and Glaser, Biochemistry 25, 878-84 (1986); PA1 Towler and Glaser, Proc. Natl. Acad. Sci. USA 83, 2812-2816 (1986); PA1 Towler et al., Proc. Natl. Acad. Sci. USA 84, 2708-2712 (1987); PA1 Towler et al., J. Biol. Chem. 262, 1030-1036 (1987); PA1 Towler et al., Ann. Rev. Biochem. 57, 69-99 (1988); PA1 Heuckeroth et al., Proc. Natl. Acad. Sci. USA 85, 8795-8799 (1988); and PA1 Heuckeroth and Gordon, Proc. Natl. Acad. Sci. USA 86, 5262-5266 (1989).
Unique synthetic peptides having relatively short amino acid sequences which are useful as substrates of myristoylating enzymes are described in U.S. Pat. Nos. 4,740,588 and 4,778,878. Examples of such peptides are: EQU Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg
and EQU Gly-Asn-Ala-Ala-Ser-Tyr-Arg-Arg.
Certain other unique synthetic peptides are inhibitors of myristoylating enzymes as described in U.S. Pat. Nos. 4,709,012 and 4,778,877.
In applications Ser. No. 07/208,192, filed Jun. 16, 1988, now abandoned, Ser. No. 07/402,094 filed Sep. 1, 1989, now U.S. Pat. No. 5,073,571, and Ser. No. 07/478,298 filed Feb. 9, 1990, now U.S. Pat. No. 5,082,967, novel fatty acid analog substrates of myristoylating enzymes are described which have potential use as antiviral, antifungal and antineoplastic agents. These substrate compounds are mono- and diheteroatom-substituted fatty acid analogs in which the heteroatoms are oxygen and/or sulfur which replace methylene (--CH.sub.2 --) groups in carbon positions 4 to 13 in the fatty acid chain of C.sub.13 -C.sub.14 fatty acids. Examples of such fatty acid analogs are: EQU 11-oxamyristic acid
and EQU 13-oxamyristic acid.
The CoA ester of these fatty acid analogs are substrates for NMT and are selectively transferred to subsets of cellular or viral N-myristoylproteins where they can alter protein function.